This study was a prospective single blind randomised controlled trial to compare the effects of rehabilitation with emphasis on retraining the vastus medialis (VMO) component of the quadriceps femoris muscle and rehabilitation with emphasis on general strengthening of the quadriceps femoris muscles on pain, function and Quality of Life in patients with patellofemoral pain syndrome (PFPS). Patients with PFPS (n = 69) were recruited from a hospital orthopaedic clinic and randomised into three groups: (1) physiotherapy with emphasis on selectively retraining the VMO (Selective); (2) physiotherapy with emphasis on general strengthening of the quadriceps femoris muscles (General); and (3) a no-treatment control group (Control). The three groups were then compared before and after an eight-week rehabilitation period. The Selective and General groups demonstrated statistically significant and 'moderate' to 'large' effect size reductions in pain when compared to the Control group. Both the Selective and General groups displayed statistically significant and 'moderate' and 'large' effect size improvements in subjective function and Quality of Life compared to the Control group. Knee flexion excursion during the stance phase of gait, demonstrated that there were no statistical significant differences and only 'trivial' to 'small' effect size differences between the Selective or General groups and the Control group. A large number of PFPS patients can experience significant improvements in pain, function and Quality of Life, at least in the short term, with quadriceps femoris rehabilitation, with or without emphasis on selective activation of the VMO component. Both approaches would seem acceptable for rehabilitating patients with PFPS. It may be appropriate to undertake exercises involving selective activation of the vastus medialis early in the rehabilitation process, however, clinicians should not overly focus on selective activation before progressing rehabilitation, especially in more chronic cases with significant participation restrictions.